Antibiotic resistance in pathogenic bacteria like Staphylococcus aureus has made it increasingly difficult to treat bacterial infections. Methicillin resistant S. aureus (MRSA) strains are resistant to -lactams and are becoming increasingly resistant to 'last resort' drugs such as vancomycin. In an effort to identify new targets for antibiotic therapy, a transposon mutagenesis screen identifying genes synthetically lethal with wall techoic acids (WTAs) was recently employed by the Walker lab. WTAs have been identified as pathogenesis factors and their inhibition sensitizes MRSA strains to -lactams, linking WTAs to cell division/peptidoglycan biosynthesis. Several genes that become essential with the chemical inhibition of WTAs were identified, including two that were previously uncharacterized. The two gene products are among 5 proteins in S. aureus belonging to the CAAX protease homolog (CPH) protein family. CAAX proteases and their homologs are ubiquitous from bacteria to humans, and their roles in modifying prenylated membrane proteins, such as the yeast GTPase Ras, have been well-characterized in eukaryotes. However, CPHs in prokaryotes have not been well-studied and their functions remain elusive. Two recent studies pointed to a role for CPHs in peptidoglycan biosynthesis and cell division. In light of the connection to peptidoglycan biosynthesis, as well as the synthetic lethal screen functionally linking WTAs to CPHs, a study of the role of CPHs in cell division/cell wall biogenesis is warranted. The objective of this proposal is to illuminate the roles of CPHs in cell division and cell wall biogenesis by: 1) elucidating genetic interactions involving CPH genes, 2) characterizing protein-interacting partners, 3) ascertaining the role of CPH proteins in pathogenesis, and 4) performing high-throughput screening to identify small molecules that are useful inhibitors of CPHs. We will utilize high-throughput and traditional genetic and biochemical techniques as well as pharmacological experiments to attain these objectives. State-of-the-art transposon mutagenesis, as well as transposon-assisted suppressor mutant screens, will be performed to elucidate interaction networks that will yield a mechanistic understanding of the genes that are required for CPH function. Additionally, biochemical assays will be utilized to determine the essential residues and domains of these CPH proteins and discover novel protein-protein interactions. Of particular importance is the identification of interaction networks between the CPHs and cell division/cell wall biogenesis machinery. Due to the synthetic lethality of WTAs and CPHs, it is expected that CPH-deleted MRSA strains are likely to be pathogenically hindered in vivo, making these proteins prime targets for a high-throughput inhibitor screen. This comprehensive approach will allow us to determine the functional linkage between CPHs and cell division/cell wall biogenesis, and will inform us of the feasibility of targeting CPHs in the treatment of antibiotic- resistant infections.